Gallo Eugenio, Jarvik Jonathan W
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
J Cell Sci. 2017 Aug 1;130(15):2644-2653. doi: 10.1242/jcs.202952. Epub 2017 Jun 14.
A novel bi-partite fluorescence platform exploits the high affinity and selectivity of antibody scaffolds to capture and activate small-molecule fluorogens. In this report, we investigated the property of multi-selectivity activation by a single antibody against diverse cyanine family fluorogens. Our fluorescence screen identified three cell-impermeant fluorogens, each with unique emission spectra (blue, green and red) and nanomolar affinities. Most importantly, as a protein fusion tag to G-protein-coupled receptors, the antibody biosensor retained full activity - displaying bright fluorogen signals with minimal background on live cells. Because fluorogen-activating antibodies interact with their target ligands via non-covalent interactions, we were able to perform advanced multi-color detection strategies on live cells, previously difficult or impossible with conventional reporters. We found that by fine-tuning the concentrations of the different color fluorogen molecules in solution, a user may interchange the fluorescence signal (onset versus offset), execute real-time signal exchange via fluorogen competition, measure multi-channel fluorescence via co-labeling, and assess real-time cell surface receptor traffic via pulse-chase experiments. Thus, here we inform of an innovative reporter technology based on tri-color signal that allows user-defined fluorescence tuning in live-cell applications.
一种新型的双组分荧光平台利用抗体支架的高亲和力和选择性来捕获和激活小分子荧光团。在本报告中，我们研究了一种针对多种花菁家族荧光团的单克隆抗体的多重选择性激活特性。我们的荧光筛选鉴定出三种细胞不可渗透的荧光团，每种都具有独特的发射光谱（蓝色、绿色和红色）和纳摩尔亲和力。最重要的是，作为一种与G蛋白偶联受体的蛋白质融合标签，抗体生物传感器保留了全部活性——在活细胞上显示出明亮的荧光团信号，背景极小。由于荧光团激活抗体通过非共价相互作用与其靶配体相互作用，我们能够在活细胞上执行先进的多色检测策略，而这在以前使用传统报告分子时是困难或不可能的。我们发现，通过微调溶液中不同颜色荧光团分子的浓度，用户可以互换荧光信号（起始与偏移），通过荧光团竞争进行实时信号交换，通过共标记测量多通道荧光，并通过脉冲追踪实验评估实时细胞表面受体运输。因此，在这里我们介绍了一种基于三色信号的创新报告技术，该技术允许在活细胞应用中进行用户定义的荧光调节。
